Presentation on theme: "Smoking Habits There are over 1 billion people in the world that smoke tobacco There are over 1 billion people."— Presentation transcript:

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Smoking Habits There are over 1 billion people in the world that smoke tobacco There are over 1 billion people in the world that smoke tobacco Of these 5-6 million will die on an annual basis Of these 5-6 million will die on an annual basis This habit increases the likelihood of developing lung cancer to 20 times that of a non-smoker This habit increases the likelihood of developing lung cancer to 20 times that of a non-smoker

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Sequencing of a SCLC cell line Why use SCLC? Why use SCLC? Not surgically resected Not surgically resected Cell line Cell line NCI-H209 NCI-H209 Immortal cell line Immortal cell line 55-year-old male with SCLC 55-year-old male with SCLC Smoking history not recorded Smoking history not recorded Showed histologically typical small cells Showed histologically typical small cells >97% of such tumors associated with tobacco smoking >97% of such tumors associated with tobacco smoking Taken before chemotherapy Taken before chemotherapy

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Each molecule attached to a bead Each molecule attached to a bead Amplified using emulsion PCR Amplified using emulsion PCR 3’ end modification 3’ end modification Beads are covalently attached to a glass slide Beads are covalently attached to a glass slide

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A universal sequencing primer, ligase, and a set of fluorescently labeled di-base probes are introduced A universal sequencing primer, ligase, and a set of fluorescently labeled di-base probes are introduced

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Multiple cycles of ligation, detection, and cleavage performed Multiple cycles of ligation, detection, and cleavage performed After the template has been read, synthesized strand removed After the template has been read, synthesized strand removed Primer attaches to template offset by 1 nucleotide Primer attaches to template offset by 1 nucleotide

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Non-synonymous vs. Synonymous Non-synonymous Non-synonymous Codes for different amino acid Codes for different amino acid Synonymous Synonymous Amino acid produced not modified Amino acid produced not modified Accumulation of mutations increasing fitness will be shown as an excess of non-synonymous Accumulation of mutations increasing fitness will be shown as an excess of non-synonymous Observed ratio not different than that expected by chance Observed ratio not different than that expected by chance Suggests that the majority of coding variants do not confer selective advantage Suggests that the majority of coding variants do not confer selective advantage

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Mutations in regulatory regions Little known about mutations occurring on either side of transcription start sites Little known about mutations occurring on either side of transcription start sites Supplementary Fig. 2A Supplementary Fig. 2A Find somatic substitutions within 2kb of known transcription start sites Find somatic substitutions within 2kb of known transcription start sites

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Apply hidden Markov models Apply hidden Markov models AI program that can be trained to find sequences AI program that can be trained to find sequences Predict which substitutions might affect transcription factor binding sites Predict which substitutions might affect transcription factor binding sites Supplementary Fig. 2B Supplementary Fig. 2B Distribution observed no different than that those mutations seen in random “simulated sets” of mutations Distribution observed no different than that those mutations seen in random “simulated sets” of mutations

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Big picture of somatic mutations Data indicates that most of the mutations in the coding and promoter regions are passenger events Data indicates that most of the mutations in the coding and promoter regions are passenger events Events that don’t contribute to the development of cancer, but have occurred during cancer growth Events that don’t contribute to the development of cancer, but have occurred during cancer growth Mutations confer no selective advantage to the cells Mutations confer no selective advantage to the cells

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The carcinogen binds to the DNA forming a bulky adducts at purine bases (guanine and adenine). -Change the alpha helix -Allow non-Watson–Crick pairing -Get in the way

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Most Common Transversions G>T/C>A (34%) G>A/C>T (21%) A>G/T>C (19%) Top 3 transversions are all purines…

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This distribution of transversions is consistent with the literature This distribution of transversions is consistent with the literature Shows there is consistenency with mutational patterns. Shows there is consistenency with mutational patterns. Control for in vivo mutation Control for in vivo mutation

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G>T transversions occur more frequently at methylated CpG dinucleotides G>T transversions occur more frequently at methylated CpG dinucleotides In mammals, 70% to 80% of CpG are methylated In mammals, 70% to 80% of CpG are methylated (34%) of total mutations

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5’ 3’ 5’ CpG Island: High frequency of cytosine connected to guanine. CpG islands are regions that contain a high CpG content. CpG islands are regions that contain a high CpG content. They are in and near approximately 40% of promoters of mammalian genes. They are in and near approximately 40% of promoters of mammalian genes.

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It’s getting complicated so lets recap: Most transversion mutations (34% of total) are G>T Most transversion mutations (34% of total) are G>T The G >T mutations happen often at CpG sites The G >T mutations happen often at CpG sites The G >T mutations which happen at CpG sites are often methylated CpG sites The G >T mutations which happen at CpG sites are often methylated CpG sites

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When looking at guanines in the genome, how often is the nucleotide preceding it a cytosine? This often in the genome, a C is expected to precede a G

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When looking at guanines in the genome, how often is the nucleotide preceding it a cytosine? This often in a G>T mutations, a C precedes the G

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Wait, what? 5’ 3’ 5’ -N-N-N-N-?-G-N-N-N-N-N-N-N-C-G-N-N-N-N-?-G>T-N-N-N-N-N-?- G-N-N-N- The expected fraction of CpG’s per Guanine in genomic DNA The fraction of G>Ts mutations on CpG’s per guanine in CpG islands. If everything was random, we would expect the G>T mutations to have an equal make up of CpG/G, as genomic CpG/G… …but that is not so!

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When looking at guanines in the genome, how often is the nucleotide preceding it a cytosine? This often in a G>T mutations, a C precedes the G

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When looking at guanines in the genome, how often is the nucleotide preceding it a cytosine? This often in a G>A mutation, a C precedes the G Often occur outside CpG islands. Often occur outside CpG islands. Unusually high fraction likely due to spontaneous deamination of methylated cytosine to thymine Unusually high fraction likely due to spontaneous deamination of methylated cytosine to thymine

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When looking at guanines in the genome, how often is the nucleotide preceding it a cytosine? This often in a G>C mutation, a C precedes the G similar to G>T but these were significantly more likely to occur within CpG islands similar to G>T but these were significantly more likely to occur within CpG islands

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WHAT DOES THIS ALL MEAN? “Thus, the sequence context of the 23,000 mutations in the NCI-H209 genome provides tremendous power to identify multiple distinctive mutation signatures, not evident from targeted re-sequencing studies of limited genomic regions.”

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It’s getting complicated (still) so lets recap: Most transversion mutations (34% of total) are G>T Most transversion mutations (34% of total) are G>T The G >T mutations happen often at CpG sites The G >T mutations happen often at CpG sites The G >T mutations which happen at CpG sites are often methylated CpG sites. The G >T mutations which happen at CpG sites are often methylated CpG sites.

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So how does the Methylation play into all this? Only 10–20% of CpG dinucleotides in CpG islands are methylated while 60–70% CpG sites are methylated outside the islands. Only 10–20% of CpG dinucleotides in CpG islands are methylated while 60–70% CpG sites are methylated outside the islands. This provides a model to see how methylation of CpG sites affects C>T mutations. This provides a model to see how methylation of CpG sites affects C>T mutations.

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Can’t we fix this??? Bulky adducts on purines are the most common source of DNA damage from tobacco carcinogens. Bulky adducts on purines are the most common source of DNA damage from tobacco carcinogens. These bulky adducts get in the way of the RNA polymerase. These bulky adducts get in the way of the RNA polymerase. When the RNA polymerase stops, it recruits nucleotide excision repair machinery, leading to excision of the altered nucleotide, preventing mutation. When the RNA polymerase stops, it recruits nucleotide excision repair machinery, leading to excision of the altered nucleotide, preventing mutation.

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The more expression, the more the repair. The more expression, the more the repair. Mutation repair in non transcribed regions occurred less frequently than transcribed regions (good!). Mutation repair in non transcribed regions occurred less frequently than transcribed regions (good!).

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This suggests at least two separate DNA repair pathways This suggests at least two separate DNA repair pathways Which suggests “distinct physicochemical effects on DNA structure, with variable recognition and excision by the genome surveillance machinery.” Which suggests “distinct physicochemical effects on DNA structure, with variable recognition and excision by the genome surveillance machinery.”

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Figure 4. NCI-H2171 & LU-135 show elevated levels of expression NCI-H2171 & LU-135 show elevated levels of expression SCLC in general have a greater normalized expression of CHD7 than non-SCLC & other tumor types

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CHD7 Summary CHD7 rearrangements found in 3 SCLC cell linesCHD7 rearrangements found in 3 SCLC cell lines LU-135 & NCI-H2171: have PVTI-CHD7 fusion genes + MYC amplification LU-135 & NCI-H2171: have PVTI-CHD7 fusion genes + MYC amplification PVTI downstream of MYC & may be a transcriptional target of the MYC protein PVTI downstream of MYC & may be a transcriptional target of the MYC protein Insertion of CHD7 with subsequent amplification results in increased gene copy number & regulatory elements Insertion of CHD7 with subsequent amplification results in increased gene copy number & regulatory elements OVEREXPRESSION OVEREXPRESSION NCI-H209: duplication of parts of the CHD7 geneNCI-H209: duplication of parts of the CHD7 gene CHD7 is a chromatin remodeller that promotes enhancer-mediated transcription through histone methylation CHD7 is a chromatin remodeller that promotes enhancer-mediated transcription through histone methylation Histone modifiers have been implicated as cancer genes previously Histone modifiers have been implicated as cancer genes previously Rearrangements of CHD7 would make for an Rearrangements of CHD7 would make for an interesting extension of this paper interesting extension of this paper

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Summary Each mutation due to the carcinogen affect causes consequences in three processes: Each mutation due to the carcinogen affect causes consequences in three processes: Chemical modification of a purine Chemical modification of a purine Failure to repair via surveillance pathways Failure to repair via surveillance pathways Incorrect nucleotide incorporation due to base distortion during DNA replication Incorrect nucleotide incorporation due to base distortion during DNA replication

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After Thought Lung cancer develops after 50 pack years of smoking Lung cancer develops after 50 pack years of smoking 7,300 cigarettes a year 7,300 cigarettes a year On average you acquire one mutation for every 15 cigarettes smoked On average you acquire one mutation for every 15 cigarettes smoked